Validation and Functional Analysis of Reference and Tissue-Specific Genes in Adipose Tissue of Freshwater Drum, Aplodinotus grunniens, under Starvation and Hypothermia Stress

Adipose tissue is critical to the growth, development, and physiological health of animals. Reference genes play an essential role in normalizing the expression of mRNAs. Tissue-specific genes are preferred for their function and expression in specific tissues or cell types. Identification of these genes contributes to understanding the tissue–gene relationship and the etiology and discovery of new tissue-specific targets. Therefore, reference genes and tissue-specific genes in the adipose tissue of Aplodinotus grunniens were identified to explore their function under exogenous starvation (1 d, 2 w, 6 w) and hypothermic stress (18 °C and 10 °C for 2 d and 8 d) in this study. Results suggest that 60SRP was the most stable reference gene in adipose tissue. Meanwhile, eight genes were validated as tissue-specific candidates from the high-throughput sequencing database, while seven of them (ADM2, β2GP1, CAMK1G, CIDE3, FAM213A, HSL, KRT222, and NCEH1) were confirmed in adipose tissue. Additionally, these seven tissue-specific genes were active in response to starvation and hypothermic stress in a time- or temperature-dependent manner. These results demonstrate that adipose-specific genes can be identified using stable internal reference genes, thereby identifying specific important functions under starvation and hypothermic stress, which provides tissue-specific targets for adipose regulation in A. grunniens.


Introduction
Adipose tissue of fish is distributed throughout the body (liver, brain, subcutaneous, abdominal cavity, and red and white muscles). Different parts of adipose tissue serve different functions, such as that in the subcutaneous and peri-insular areas, which influences the carcass and fillet yields, and in the muscle depots, which regulates the organoleptic quality of the meat [1]. Adipose tissue regulates systemic metabolic homeostasis through its profound effects on energy storage, endocrine function, and adaptive thermogenesis. When the feed intake exceeds their expenditure, excessive energy will converse into triglycerides and store in the body as fatty acids, which can in turn serve as an energy supply under feed restriction [2]. As an endocrine organ, adipose tissue functions importantly in nutritional, neurological, hormonal, and metabolic regulation by synthesizing bioactive compounds and regulating metabolic homeostasis [3], such as triglyceride synthesis and storage, adipokine secretion, and overall energy balance [4,5].
Generally, fats in the body are deposited in adipose tissue. At present, most economically important fish are fed with compound diets, which are generally characterized by 2 of 16 energy surpluses and nutrient imbalances. Similarly with mammals, excessive fat accumulation in fish represents a disturbed metabolic condition that not only damages the health of the fish but also reduces the quality of the product [6,7]. Meanwhile, excessive lipid deposition also negatively alters the morphology of tissues or cells, resulting in a variety of negative effects on fish health [8,9]. Therefore, reducing excessive lipid deposition contributes to improving the health and quality of aquatic products. Feed restriction has been shown to reduce abdominal fat accumulation [10,11]. During this process, exogenous food intake was restricted, while the endogenous substances were active to generate energy for the maintenance of normal physiological activity, such as carbohydrates, lipids, and proteins [12][13][14]. Research in Danio rerio revealed that carbohydrate is principally used for energy supply during the early stage of starvation (within 24 h), while fat is mobilized for energy supply during 2~4 d of starvation. However, protein is barely expended for energy supply during short-term starvation [15]. Therefore, short-term feed restriction is an effective approach to revealing the lipid metabolism of animals.
The growth, physiology, and reproduction of fish are greatly affected by the ambient temperature [16]. When temperature changes exceed the adaptive range of aquatic animals, hypoor hyperthermic stress can cause metabolic disorders, tissue damage, and even death [17,18]. Under hypothermia, aquatic animals unconsciously consume more endogenous energy substances to increase energy production. Several studies indicate that lipid metabolism functions importantly in hypothermia resistance. Specifically, to cope with hypothermia, fish will alter their lipid metabolism. In order to compensate for the effects on enzyme function, fish will increase their membrane fluidity in response to the drop in ambient temperature [19][20][21][22]. Changes in phospholipid composition and cholesterol content [23] are effective ways to accomplish this. In response to hypothermic stress, fish can change the fatty acid composition of various tissue cell membranes by controlling the expression of enzymes and genes implicated in fatty acid metabolism [24]. Therefore, lipid metabolism in adipose tissue can alleviate the side effects of hypothermic stress and protect the health of the body.
Tissue-specific genes are a class of genes with specific functions in particular tissues or cells [25]. Identifying the function of these genes provides a better understanding of tissue genetic relationships, which can contribute to the discovery of new tissue-specific targets. In aquatic animals, tissue-specific genes have also been identified in some species, such as ovarian development-related genes in Macrobrachium nipponense [26], growth and feedingrelated genes in Aristichthys nobilis [27] and immune-related genes in Cyprinus carpio [28]. In this paper, adipose-specific genes were identified, and the molecular regulation mechanisms in lipid metabolism were investigated in freshwater drums.
Freshwater drum (Aplodinotus grunniens) belongs to Aplodinotus and inhabits only freshwater during its lifetime [29]. In the aspect of edibility, freshwater drum features high edible proportions and delicious and nutritious flesh. With these prospects, we imported freshwater drum larvae and achieved a worldwide breakthrough in the advanced research of artificial breeding, feeding and domestication. In 2022 we achieved large-scale fry breeding, laying the groundwork for the industrialized development of freshwater drum culture and the breeding of new varieties [30][31][32][33]. However, the molecular mechanisms of adipose tissue in freshwater drums are hitherto unknown. Therefore, it is necessary to screen out the target molecules of adipose tissue, to understand the role of adipose tissue in freshwater drum development, and contribute to subsequent functional genomics. Therefore, the above updated research progress inspired us to conduct this study to identify the stable internal reference genes, thereby validating the tissue-specific genes that function importantly in the adipose tissue of freshwater drum. Consecutively, we also validated whether these tissue-specific genes function importantly with starvation and hypothermic stress, which could induce metabolic and adaptive physiological dysfunction of adipose tissue. These results are novel in understanding the regulation mechanism of adipose tissue in freshwater drums. At the same time, these vital genes may also serve as candidate targets for starvation and hypothermia resistance, which are conducive to the sustainable development of freshwater drum.

Ethics Statement
This research has received approval from the Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, China). All animal procedures were performed in accordance with the Guideline for the Care and Use of Laboratory Animals in China.

Experimental Animals and Experimental Design
The laboratory fish in the present study were the first-generation larvae of freshwater drums introduced from the United States. The experiment was carried out in an indoor temperature-adjustable circular aquaculture system (specifications for ϕ 820 mm × 700 mm, 300 L) of the Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences. Water was obtained from a deep well and aerated before use. Before the experiments, the fish were acclimated in a water tank at 25 • C for 14 days. The fish were fed with fresh bait twice daily (8:00 and 16:00, 3-5% weight).
For the starvation experiment, a one-factor, completely randomized experiment was designed. Four treatments were established, starvation for 0 d was set as the control, as followed by starvation for 1 d, 2 w, and 6 w. Two hundred and forty fish with an average body weight of 20.88 ± 2.75 g were divided into four groups (three tanks per group, 20 individuals per tank). During the experiment, all fish were not fed.
For the hypothermia experiment, a two-factor, completely randomized experiment was designed. Specifically, 25 • C was set as the control, 18 • C and 10 • C were set as hypothermia treatment for 2 d and 8 d, and six treatments were finally established (Con-2 d, LT18-2 d, LT10-2 d, Con-8 d, LT18-8 d, and LT10-8 d). One hundred and eighty fish were randomly divided into three groups (three tanks per group, 20 individuals per tank). The water temperature was decreased gradually from 25 • C to 18 • C and 10 • C in 15 h at a rate of 1 • C/h in the recirculating aquaculture system and maintained for eight days. During the hypothermia experiment, fish were fed as previously.
In this study, starvation and hypothermic stress experiments were performed as two independent experiments. Throughout all experiments, the water parameters were kept as follows: dissolved oxygen > 6 mg L −1 , pH 7.2~7.8, NO 2 − < 0.02 mg L −1 , and NH 3 < 0.05 mg L −1 .

Sample Collection
The starvation experiment samples were collected at 0 d (control), 1 d, 2 w, and 6 w. Three fish were collected per tank at each time point for a total of nine biological replicates. The hypothermia experimental samples were collected at 2 d and 8 d at 25 • C (control), 18 • C, and 10 • C, respectively. At each temperature, three fish were collected from each replicate at each time point, for a total of nine biological replicates. In the hypothermic stress experiment, the fish were starved for 24 h to evacuate the alimentary tract contents before collecting samples. In the process of sampling, fish from each tank were randomly taken and anesthetized with MS-222 (100 mg/L), after which the abdominal fat (AF) was collected, immediately frozen in liquid nitrogen, and stored at −80 • C. Meanwhile, 15 tissues (including the brain (BR), head kidney (HK), trunk kidney (TK), liver (LI), foregut intestine (FI), mid-intestine (MI), hind intestine (HI), muscles (MU), abdominal fat (AF), gill (GI), spleen (SP), heart (HE), skin (SK), stomach (ST), and testis (TE)) from the fish reared at 25 • C were collected to conduct tissue-specific and internal reference gene analyses.

Extraction of Total RNA and Synthesis of cDNA
Total RNA was extracted from the tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA quality and concentration were determined by A260/280 and an ultra-micro spectrophotometer, respectively. Subsequently, cDNA was synthesized with PrimeScript TM RT Master Mix reverse transcription kit (Takara, Dalian, China). The above operations were carried out according to the instruction manual.

RT-PCR Analysis
Based on the mRNA sequences obtained from the transcriptome database, the primers were designed with Primer Premier 5.0. All the primers (shown in Table 1) were synthesized by Shanghai Generay Biotechnology, Co., Ltd., China. According to the manufacturer's protocol, RT-PCR was performed with SYBR Green (Takara, Dalian, China) on Takara 800 Fast RT-PCR System.

Statistical Analysis
In the study, all data were calculated using SPSS software (version 26.0) and presented as mean ± standard error mean (SEM). The most stable internal reference genes were screened by ∆CT, GeNorm, NormFinder, Bestkeeper, and RefFinder. For RNA expression analysis, 2 −∆∆CT method was applied. For statistical difference evaluation, data was analyzed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test when data was under normal distribution and homoscedasticity, or else using a nonparametric test (Kruskal-Wallis test). In the starvation experiment, Students' t-test was used to analyze the difference between different starvation times. In the hypothermia experiment, two-way ANOVA was used to verify the interaction between time and temperature. To evaluate the impact of different temperatures and stress times on the expression of tissue specific genes, Student's t-test and one-way ANOVA was applied, respectively. Pearson's correlation analysis was used to assess the relevance of genes under starvation and hypothermic stress. In general, p < 0.05 was considered to be a significant difference or correlation, while p < 0.01 was regarded as an extremely significant difference or correlation.

RNA Quality and Primer Amplification Assessment
The A260/280 ratio was 1.98 ± 0.09, confirming that the RNA was pure and proteinfree. Meanwhile, the RNA integrity number (RIN) was 9.7 ± 0.2, indicating that the RNA integrity was stabilized and reliable for RT-PCR analysis ( Figure S1). Additionally, the amplification efficiency was between 95% and 108% (Table 1), and the coefficient of determination (R 2 ) for each gene ranged from 0.9951 to 0.9998 ( Figure 1A-K), which indicated that the primers were suitable for RT-PCR analysis.

Expression Ranges and Stability of Candidate Reference Genes
Next, we determined the expression of candidate reference genes in terms of CT values by RT-PCR. The results show that the CT values of the 11 internal reference genes

Expression Ranges and Stability of Candidate Reference Genes
Next, we determined the expression of candidate reference genes in terms of CT values by RT-PCR. The results show that the CT values of the 11 internal reference genes ranged from 15.05 to 35.5 ( Figure 2A). However, the CT value of SEC62 reached outside the valid range of detection, implying low mRNA abundance in tissues, and indicating that it was unsuitable as a reference gene for freshwater drum.
∆CT analysis revealed that 60SRP was frequently associated with the least deviation, implying that it has low variability compared to the other ten genes ( Figure 2B-M). GeNorm analysis demonstrated that 60SRP, RPS4, RPl7, β-actin, and EF1α exhibited high expression stability, as identified by the M value (M < 1.5, Figure 3A). NormFinder analysis ( Figure 3B) confirmed that 60SRP had the highest expression stability among the 11 reference genes. Bestkeeper can calculate the standard deviation (SD) and coefficient of variation (CV). The SD indicated that only RPS4 and β-actin can be used as reference genes (SD < 1, Figure 3C), and the CV indicated that RPS4 had the smallest coefficient of variation and better stability (Table S1). RefFinder analysis showed that 60SRP was the most stable reference gene ( Figure 3D).

Identification and Expression of Adipose Tissue-Specific Genes
Based on the above studies, tissue-specific genes were validated in freshwater drum adipose tissue using stable internal reference genes. According to the transcriptome database of 11 different tissues in freshwater drums, a total of 9 tissue-specific genes were selected (fold change > 10, p < 0.05; shown in Figure 4). Moreover, the expression of derlin-2-like (DERL2) in AF exhibited no significant difference with MI and SK (KW, p > 0.05), while the other candidate genes in AF exhibited a remarkable difference with all the other tissues (ANOVA, p < 0.01). Therefore, adrenomedullin 2 (ADM2), β2-glycoprotein I (β 2 GP1), calcium/calmodulin-dependent protein kinase type 1G (CAMK1G), keratin-like protein KRT222 (KRT222), cell death activator CIDE3-like (CIDE3), hormone-sensitive lipase (HSL), neutral cholesterol ester hydrolase 1 (NCEH1), and redox-regulatory protein (FAM213A) were identified as the candidate adipose tissue-specific genes.

Expression of Adipose Tissue-Specific Genes in Different Tissues
Next, we used RT-PCR to further verify whether the candidate genes were specifically expressed in adipose tissue. The amplification efficiency and coefficient of determination (R 2 ) of the tissue-specific genes were determined. Results show that the amplification efficiencies were between 92% and 111% (Table 1), and the R 2 for each gene ranged from 0.9541 to 0.9988 ( Figure S2), indicating that the primers of tissue-specific genes were suitable for RT-PCR experiments.

Expression of Adipose Tissue-Specific Genes in Different Tissues
Next, we used RT-PCR to further verify whether the candidate genes were specifically expressed in adipose tissue. The amplification efficiency and coefficient of Different letters indicate marked differences between AF and the other 10 tissues (p < 0.05), results were indicated as mean ± SEM or medians ± interquartile range; n = 3.

Expression Characteristics of Adipose Tissue-Specific Genes under Starvation
To further validate the role of these tissue-specific genes, we next conducted starvation stress to block food intake and improve the depletion of deposited fats in AF tissue. Results show that, compared with the control, β 2 GP1 was upregulated dramatically during starvation (T, p < 0.001). Similarly, CAMK1G at 2 w was distinctly higher than the control, 6 w (T, p < 0.001), and 1 d (T, p = 0.019). The transcriptional expression of FAM213A and NCEH1 at 1 d was significantly higher than the control (T, p < 0.01), of which FAM213A was extremely significantly downregulated at 2 w and 6 w (T, p < 0.01), Likewise, NCEH1 was significantly reduced at 2 w (T, p = 0.017) and 6 w (T, p < 0.01). HSL at 1 d and 2 w were significantly different from the control and 6 w (T, p < 0.01). The expression of KRT222 was increased at 1d of starvation compared to other times (T, p < 0.01). CIDE3 reduced as starvation duration increased, and the difference was significant only after 2 w (T, p = 0.017) and 6 w (T, p = 0.045, Figure 6). genes were suitable for RT-PCR experiments.

Expression Characteristics of Adipose Tissue-Specific Genes under Starvation
To further validate the role of these tissue-specific genes, we next conducted starvation stress to block food intake and improve the depletion of deposited fats in AF tissue. Results show that, compared with the control, β2GP1 was upregulated dramatically during starvation (T, p < 0.001). Similarly, CAMK1G at 2 w was distinctly higher than the control, 6 w (T, p < 0.001), and 1 d (T, p = 0.019). The transcriptional expression of FAM213A and NCEH1 at 1 d was significantly higher than the control (T, p < 0.01), of which FAM213A was extremely significantly downregulated at 2 w and 6 w (T, p < 0.01), Likewise, NCEH1 was significantly reduced at 2 w (T, p = 0.017) and 6 w (T, p < 0.01). HSL at 1 d and 2 w were significantly different from the control and 6 w (T, p < 0.01). The expression of KRT222 was increased at 1d of starvation compared to other times (T, p < 0.01). CIDE3 reduced as starvation duration increased, and the difference was significant only after 2 w (T, p = 0.017) and 6 w (T, p = 0.045, Figure 6). Data were analyzed by Student's t-test. * Refers to a significant difference between starvation times (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****. p < 0.0001), results were indicated as mean ± SEM; n = 9.

Expression Characteristics of Adipose Tissue-Specific Genes under Hypothermia
The key role played by tissue-specific genes was further validated under hypothermic stress in order to comprehend how hypothermia stimulates lipolysis and thermogenesis, as shown in Figure 7. At LT-18, there was no significant difference in gene expression at different times (T, p > 0.05). At LT-10, only β2GP1 significantly decreased with time increases (T, p < 0.01), whereas CAMK1G dramatically increased (T, p < 0.01). At 2 d, all genes were markedly increased at LT-18 (ANOVA, p < 0.05). As the temperature decreased, the expressions of CAMK1G, NCEH1, and KRT222 were not dramatically different from the

Expression Characteristics of Adipose Tissue-Specific Genes under Hypothermia
The key role played by tissue-specific genes was further validated under hypothermic stress in order to comprehend how hypothermia stimulates lipolysis and thermogenesis, as shown in Figure 7. At LT-18, there was no significant difference in gene expression at different times (T, p > 0.05). At LT-10, only β2GP1 significantly decreased with time increases (T, p < 0.01), whereas CAMK1G dramatically increased (T, p < 0.01). At 2 d, all genes were markedly increased at LT-18 (ANOVA, p < 0.05). As the temperature decreased, the expressions of CAMK1G, NCEH1, and KRT222 were not dramatically different from the control (ANOVA, p > 0.05). At 8 d, all genes were significantly increased at LT-18 (ANOVA, p < 0.05) but β2GP1, NCEH1, KRT222, and CIDE3 returned to their original levels at LT-10 (ANOVA, p > 0.05). Interaction analysis showed that β2GP1, HSL, and CIDE3 were affected by the interaction between time and temperature (ANOVA, p < 0.05).  Data were analyzed by one-way ANOVA, two-way ANOVA and t-test. Different letters indicate significant differences between the control group and the experimental group (p < 0.05), lowercase letters represent significant differences at 2 d and uppercase letters represent significant differences at 8 d. Asterisk Refers to a significant difference between different times at the same temperature level (** p < 0.01), results were indicated as mean ± SEM; n = 9.

Comprehensive Analysis of Tissue-Specific Genes under Hypothermia and Starvation
Based on the aforementioned information, the important role of tissue-specific genes in exogenous stress was comprehensively analyzed. The results of the Pearson correlation analysis are shown in Figure 8. We discovered that starvation stress and hypothermic stress mainly affected the expression of β 2 GP1, FAM213A, HSL, KRT222, and CIDE3 genes, while there was no significant correlation increase in CAMK1G and NCFH1. At 2 d of short-term hypothermia, only FAM213A was significantly correlated at 6 w of starvation. At 8 d of prolonged hypothermia, starvation for 1 d resulted in increases in β 2 GP1 and FAM213A, and starvation for 2 w resulted in increases in HSL, KRT222, and CIDE3. analysis are shown in Figure 8. We discovered that starvation stress and hypothermic stress mainly affected the expression of β2GP1, FAM213A, HSL, KRT222, and CIDE3 genes, while there was no significant correlation increase in CAMK1G and NCFH1. At 2 d of short-term hypothermia, only FAM213A was significantly correlated at 6 w of starvation. At 8 d of prolonged hypothermia, starvation for 1 d resulted in increases in β2GP1 and FAM213A, and starvation for 2 w resulted in increases in HSL, KRT222, and CIDE3.

Discussion
Suitable internal reference genes could improve the reliability of RT-PCR results, thereby confirming the expression and function of specific genes. In summary, there is no common internal reference gene available for gene expression standardization under all conditions [34,35]. It is generally known that β-actin and GAPDH are the most used reference genes. However, it was found that β-actin and GAPDH exhibit remarkable differences in different tissues and experimental conditions [36][37][38]. Similarly, it was demonstrated in the present study that GAPDH and β-actin are not applicable as reference genes for adipose tissue in freshwater drum, which may be related to their functions. β-actin is an essential component of cell structure maintenance, cell motility, cytoplasmic division, endocytosis, and cell adhesion [39], while it has been found that β-actin is differentially expressed at different stages of preadipocyte differentiation [40]. GAPDH plays a vital role in glycolytic processes [41] and has also been shown to be unsuitable as a common reference gene in adipose tissue due to its differential expression levels in brown adipose tissue (BAT) and white adipose tissue (WAT) [42]. In addition to the above-mentioned classical internal reference genes, many studies have also found that RPL7 [43,44] and Rpl13α [45,46] can also be used as internal references. In the present study, 60SRP was the most stably expressed gene in freshwater drum adipose tissue. As a member of the ribosomal protein family, 60SRP plays an essential role in protein synthesis, cell proliferation,

Discussion
Suitable internal reference genes could improve the reliability of RT-PCR results, thereby confirming the expression and function of specific genes. In summary, there is no common internal reference gene available for gene expression standardization under all conditions [34,35]. It is generally known that β-actin and GAPDH are the most used reference genes. However, it was found that β-actin and GAPDH exhibit remarkable differences in different tissues and experimental conditions [36][37][38]. Similarly, it was demonstrated in the present study that GAPDH and β-actin are not applicable as reference genes for adipose tissue in freshwater drum, which may be related to their functions. β-actin is an essential component of cell structure maintenance, cell motility, cytoplasmic division, endocytosis, and cell adhesion [39], while it has been found that β-actin is differentially expressed at different stages of preadipocyte differentiation [40]. GAPDH plays a vital role in glycolytic processes [41] and has also been shown to be unsuitable as a common reference gene in adipose tissue due to its differential expression levels in brown adipose tissue (BAT) and white adipose tissue (WAT) [42]. In addition to the above-mentioned classical internal reference genes, many studies have also found that RPL7 [43,44] and Rpl13α [45,46] can also be used as internal references. In the present study, 60SRP was the most stably expressed gene in freshwater drum adipose tissue. As a member of the ribosomal protein family, 60SRP plays an essential role in protein synthesis, cell proliferation, apoptosis, multiple regulations of development, and malignant transformation. It has been shown that 60SRP can serve as an internal reference gene for fish tissues [47], which has the same results as the present study.
As an endocrine organ, adipose tissue is critical for a variety of physiological functions such as postprandial uptake of glucose and fatty acids, appetite control, and insulin sensitivity [60]. Dysfunction of adipose tissue will induce metabolic dysregulation, which in turn inhibits substrate uptake, secretory profile alteration, angiogenesis stimulation, and inflammatory cell recruitment [60]. Therefore, maintaining a relatively stable state and function of adipose tissue is vital for the health of animals. Next, we further evaluate the function of the adipose tissue-specific genes with adverse stress that could impair the function of adipose tissue. As prevalent extrinsic variables, starvation and hypothermia have a huge impact on lipid metabolism. Studies have shown that both hypothermia and starvation lead to impaired triglyceride metabolic processes [61,62]. Hypothermic stress was found to have a facilitative effect on lipid metabolism in species such as C. carpio [63], Sparus aurata [64], D. rerio [15]. Starvation stress was also found to consume body fat in species such as Pseudobagrus vachelli [65], Anguilla anguilla [66], Gadus morhua and Esox lucius [67]. Similarly, we found that CIDE3 levels were decreasing under prolonged LT-10 as well as starvation, indicating that starvation stress and prolonged cold stimulation cause a decrease in the rate of lipid synthesis and accumulation. The significant increase in apolipoprotein β 2 GP1, lipolytic genes HSL and NCEH1 under LT-18, and starvation indicated that the process of lipolytic translocation was accelerated, and the organism also promoted cell proliferation and differentiation by increasing the level of CAMK1G. These results suggest that promoting lipolytic metabolism is one of the most important ways to ensure energy supply in fish. In addition, fish organism cells transmit the hypothermia signal to the nucleus through various stress pathways, initiating the hypothermic stress response, and establishing new intracellular homeostasis after sensing a hypothermia stimulus [68]. For instance, the fish central nervous system is stimulated by hypothermia to activate ion channels as hypothermia receptors during hypothermic stress, which excites the inward flow of extracellular calcium ions and related kinases to implement the transmission of hypothermia signals [68]. Since CAMK1G can release calcium ions, this may also be one of the reasons for its increase at LT-18 and decrease at LT-10. Extensive studies showed that changes in environmental conditions could disrupt the antioxidant defense system in aquatic animals, resulting in stress responses [69][70][71]. In response to detrimental external environments, we found that freshwater drum protected the organism from oxidative stress by increasing the expression of FAM213A and KRT222. The above results indicate that both hypothermia and starvation may lead to molecular, cellular, and tissue damage. However, a distinct result showed that the expression of nearly all genes was reduced during prolonged starvation and hypothermia, indicating that the regulatory ability of these genes in fish is not unlimited. If the stimulus exceeds a certain intensity, it will cause structural damage to tissues or cells, reducing the fish's ability to self-regulate. However, lipid transport β 2 GP1 and lipolysis HSL genes are still increased during prolonged starvation compared to the control, which indicates that fish will reduce their metabolic level to conserve energy. Furthermore, fish continue to keep their metabolism above a certain level as much as possible to ensure normal life activities [72]. Additionally, through correlation analysis of starvation and hypothermia, we found a remarkable positive correlation between starvation and prolonged hypothermia. The above results demonstrate that in the early stages of starvation and short-term hypothermia, the body's energy requirements are relatively low, and energy metabolism and resistance to external environmental changes may be altered by increasing β 2 GP1 and FAM213A. As starvation prolongs and temperature decreases, the body regulates its metabolic rate to supply life-sustaining caloric energy and protect the body's health, mainly by altering the expression of FAM213A, HSL, KRT222, and CIDE3.
The above findings indicate the sensitivity of freshwater drums to environmental influences. When they are under stress, their normal metabolic processes may not provide sufficient energy, therefore mobilizing adipose tissue to take part in the regulation of energy homeostasis. The adipose-specific genes identified in this paper (β 2 GP1, CAMK1G, CIDE3, HSL, NCEH1, KRT222, and FAM213A) may play different essential roles in protecting the organism from exogenous stress and in promoting lipolysis to provide energy to the organism, and it is speculated that these genes may be key regulators of adipose tissue in response to exogenous stress states.

Conclusions
In this study, we identified a stable internal reference gene (60SRP) in adipose tissue and validated adipose tissue-specific genes by using 60SRP. We also verified the important functions of these specific genes under starvation stress and hypothermic stress. For a variety of roles, β 2 GP1 in lipid transport; CAMK1G in adipocyte proliferation and differentiation; CIDE3 in lipid synthesis; HSL, NCEH1 in lipolysis for energy supply; KRT222, FAM213A in protecting adipose cell structure against exogenous stress. These results provide tissue-specific targets for the mechanisms of lipid regulation in freshwater drums under exogenous stress, which could contribute to the development of freshwater drum culture.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/cells12091328/s1, Figure S1: RNA integrity number by RNA 6000 Nano LabChip for the samples; Figure S2: Standard curve of tissue-specific candidate genes in A. grunniens; Table S1: Bestkeeper Standard Deviation and Coefficient of Variation.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to being involved in another unpublished project.